N-methyl-D-aspartate receptors (NMDARs) play a crucial role in cognition and long-term memory, and have been implicated in a variety of pathological conditions, including stroke, intractable pain, neurodegenerative diseases, and spinal cord injury. We have shown that endogenous neurosteroids, such as pregnenolune sulfate (PS), can specifically modulate NMDA receptor function. Evidence for at least two neurosteroid recognition sites has emerged, one associated with positive modulation and the other with negative modulation. We have demonstrated recently that the direction of modulation by PS is dependent upon which of the NMDA receptor Type-2 subunits is present. NR2A or NR2B confers PS potentiation, whereas NR2C or NR2D confers PS inhibition. To define the structural determinants of receptor subtype-specific neurosteroid modulation, multiple chimeras from NR2B and NR2D parent cDNAs were constructed and analyzed by voltage clamp electrophysiology in Xenopus oocytes containing transiently expressed receptors. We have identified a Steroid Modulatory Domain 1 (SMD1) on NR2B, containing the fourth transmembrane segment (M4) and part of the adjacent M3-M4 linker region. SMD1 is required for rapid potentiation of the NMDA response by PS. In addition to the rapid direct modulation of NMDAK by neurosteroids, there is a slow phase of modulation that is mediated by PKC. The proposed studies build on and extend these findings. [unreadable] [unreadable] Using site-directedmutagenesis of NR2B/2D and NR2D/2B chimeric subunits to eliminate and then reinstate PS modulation, specific residues that contribute to the response at different NMDAR subtypes will be identified. Cysteine substitution and sulflaydryl-modifying reagents will be used to determine whether these residues are solvent accessible and participate inPS action. Results of these studies will be used to formulate a pharmacological model of NMDAR modulation by neurosteroids. In addition, the possible interactions between the steroid, zinc, and proton modulatory sites will beinvestigated. We will also investigate the mechanism of slow potentiation by PS, which, in contrast to rapid potentiation, requires the action of cellular signal-transduction systems. We will determine whether the domain(s) that control slow potentiation are the same or different from those that control fast potentiation, and will investigate the contributions of rapid and slow potentiation to enhancement of excitotoxicity by PS in neurons and transfected HEK cells. These studies will enhance our understanding of the structural basis that underlies the specificity of neurosteroid modulation at NMDAP, s, and will provide the tools with which to study the biological and pharmacological relevance of endogenous neurosteroids in the nervous system. [unreadable] [unreadable]